Antibodies to human Elk, a voltage-gated potassium channel subunit

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of hElk, antibodies to hElk, methods of detecting hElk, methods of screening for voltage-gated potassium channel activators and inhibitors using biologically active hElk, and kits for screening for activators and inhibitors of voltage-gated potassium channels comprising hElk.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 10/160,224, filed May28, 2002, now U.S. Pat. No. 6,833,440, which is a division of U.S. Ser.No. 09/343,494, filed Jun. 30, 1999, now U.S. Pat. No. 6,413,741, whichclaims the benefit of U.S. Ser. No. 60/091,469, filed Jul. 1, 1998, andU.S. Ser. No. 60/116,621, filed Jan. 21, 1999, all of which are hereinincorporated by reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences ofhElk, antibodies to hElk, methods of detecting hElk, methods ofscreening for voltage-gated potassium channel activators and inhibitorsusing biologically active hElk, and kits for screening for activatorsand inhibitors of voltage gated potassium channels comprising hElk.

BACKGROUND OF THE INVENTION

Potassium channels are involved in a number of physiological processes,including regulation of heartbeat, dilation of arteries, release ofinsulin, excitability of nerve cells, and regulation of renalelectrolyte transport. Potassium channels are thus found in a widevariety of animal cells such as nervous, muscular, glandular, immune,reproductive, and epithelial tissue. These channels allow the flow ofpotassium in and/or out of the cell under certain conditions. Forexample, the outward flow of potassium ions upon opening of thesechannels makes the interior of the cell more negative, counteractingdepolarizing voltages applied to the cell. These channels are regulated,e.g., by calcium sensitivity, voltage-gating, second messengers,extracellular ligands, and ATP-sensitivity.

Potassium channels are made by alpha subunits that fall into 8 families,based on predicted structural and functional similarities (Wei et al.,Neuropharmacology 35 (7):805-829 (1997)). Three of these families (Kv,Eag-related, and KQT) share a common motif of six transmembrane domainsand are primarily gated by voltage. Two other families, CNG and SK/IK,also contain this motif but are gated by cyclic nucleotides and calcium,respectively. The three other families of potassium channel alphasubunits have distinct patterns of transmembrane domains. Slo familypotassium channels, or BK channels have seven transmembrane domains(Meera et al., Proc. Natl. Acad. Sci. U.S.A. 94 (25):14066-71 (1997))and are gated by both voltage and calcium or pH (Schreiber et al., J.Biol. Chem. 273:3509-16 (1998)). Another family, the inward rectifierpotassium channels (Kir), belong to a structural family containing 2transmembrane domains (see, e.g., Lagrutta et al., Jpn. Heart. J.37:651-660 1996)), and an eighth functionally diverse family (TP, or“two-pore”) contains 2 tandem repeats of this inward rectifier motif.

Potassium channels are typically formed by four alpha subunits, and canbe homomeric (made of identical alpha subunits) or heteromeric (made oftwo or more distinct types of alpha subunits). In addition, potassiumchannels made from Kv, KQT and Slo or BK subunits have often been foundto contain additional, structurally distinct auxiliary, or beta,subunits. These beta subunits do not form potassium channels themselves,but instead they act as auxiliary subunits to modify the functionalproperties of channels formed by alpha subunits. For example, the Kvbeta subunits are cytoplasmic and are known to increase the surfaceexpression of Kv channels and/or modify inactivation kinetics of thechannel (Heinemann et al., J. Physiol. 493:625-633 (1996); Shi et al.,Neuron 16 (4):843-852 (1996)). In another example, the KQT family betasubunit, minK, primarily changes activation kinetics (Sanguinetti etal., Nature 384:80-83 (1996)).

The Kv superfamily of voltage-gated potassium channels includes bothheteromeric and homomeric channels that are typically composed of foursubunits. Voltage-gated potassium channels have been found in a widevariety of tissues and cell types and are involved in processes such asneuronal integration, cardiac pacemaking, muscle contraction, hormonesection, cell volume regulation, lymphocyte differentiation, and cellproliferation (see, e.g., Salinas et al., J. Biol. Chem. 39:24371-24379(1997)).

A family of voltage-gated potassium genes, known as the “Eag” or ether àgo-go family, was identified on the basis of a Drosophila behavioralmutation with a leg-shaking phenotype (see, e.g., Warmke & Ganetzky,Proc. Nat'l Acad. Sci. USA 91:3438-3442 (1994)). Family members fromDrosophila and vertebrates have been cloned and fall into threesubfamilies. One such subfamily is called the Eag subfamily and isrepresented, e.g., by Drosophila Eag (Warmke et al., Science252:1560-1562 (1991); Bruggemann et al., Nature 365:445-447 (1993)), andrat, mouse, human, and bovine Eags (Ludwig et al., EMBO J. 13:4451-4458(1994); Robertson et al. Neuropharmacology 35:841-850 (1996); Occhiodoroet al., FEBS Letters 434:177-182 (1998); Shi et al., J. Physiol.115.3:675-682 (1998); Frings et al., J. Gen Physiol. 111:583-599(1998)). A second subfamily, the Erg or “Eag-related gene” family isrepresented, e.g., by human erg (Shi et al., J. Neurosci. 17:9423-9432(1997)). Finally, a third subfamily, the Elk or “Eag-like K⁺ gene” isrepresented, e.g., by Drosophila Elk (Warmke et al., Proc. Natl. Acad.Sci. 91:3438-3442 (1994)).

SUMMARY OF THE INVENTION

The present invention thus provides for the first time human Elk, apolypeptide monomer that is an alpha subunit of an voltage-gatedpotassium channel. hElk has not been previously cloned or identified,and the present invention provides the nucleotide and amino acidsequence of hElk. Furthermore, the gene encoding hElk has been mapped tochromosome 12.

In one aspect, the present invention provides an isolated nucleic acidencoding a polypeptide monomer comprising an alpha subunit of apotassium channel, the polypeptide monomer: (i) having the ability toform, with at least one additional Elk alpha subunit, a potassiumchannel having the characteristic of voltage gating; (ii) having amonomer P-S6 region that has greater than 80%, preferably 85, 90, or 95%amino acid sequence identity to an hElk P-S6 region; and (iii)specifically binding to polyclonal antibodies generated against SEQ IDNO:1.

In another embodiment, the present invention provides an isolatednucleic acid encoding a polypeptide monomer comprising an alpha subunitof a potassium channel, the polypeptide monomer: (i) having the abilityto form, with at least one additional Elk alpha subunit, a potassiumchannel having the characteristic of voltage gating; (ii) having anextended P-S6 region that has greater than 80%, preferably 85, 90, or95% amino acid sequence identity to an hElk extended P-S6 region; and(iii) specifically binding to polyclonal antibodies generated againstSEQ ID NO:1.

In one embodiment, the nucleic acid encodes human Elk. In anotherembodiment, the nucleic acid encodes SEQ ID NO:1. In another embodiment,the nucleic acid has a nucleotide sequence of SEQ ID NO:2.

In one embodiment, the nucleic acid is amplified by primers thatselectively hybridize under stringent hybridization conditions to thesame sequence as the primer sets selected from the group consisting of:

ATGCCGGCCATGCGGGGCCTCCT, (SEQ ID NO:3) AGATGGCAGCACACCTGGCAACGCTG (SEQID NO:4) and GCCCATCTGCTGAAGACGGTGCGC, (SEQ ID NO:5)CGAAGCCCACGCTGGTGAGGCTGCTG. (SEQ ID NO:6)

In one embodiment, the nucleic acid encodes a polypeptide monomer havinga molecular weight of between about 120 kDa to about 130 kDa. In anotherembodiment, the polypeptide monomer comprises an alpha subunit of ahomomeric voltage-gated potassium channel. In another embodiment, thepolypeptide monomer comprises an alpha subunit of a heteromericvoltage-gated potassium channel.

In another embodiment, the nucleic acid selectively hybridizes undermoderately stringent hybridization conditions to a nucleotide sequenceof SEQ ID NO:2.

In another aspect, the present invention provides an isolated nucleicacid encoding a polypeptide monomer, wherein the nucleic acidspecifically hybridizes under highly stringent conditions to SEQ IDNO:2.

In another aspect, the present invention provides an isolatedpolypeptide monomer comprising an alpha subunit of a potassium channel,the polypeptide monomer: (i) having the ability to form, with at leastone additional Elk alpha subunit a potassium channel having thecharacteristic of voltage gating; (ii) having a monomer P-S6 region thathas greater than 80% amino acid sequence identity to an hElk P-S6region; and (iii) specifically binding to polyclonal antibodiesgenerated against SEQ ID NO:1.

In one embodiment, the polypeptide monomer has an amino acid sequence ofhuman Elk. In another embodiment, the polypeptide monomer has an aminoacid sequence of SEQ ID NO:1.

In another aspect, the present invention provides an antibody thatselectively binds to the polypeptide monomer described above.

In another aspect, the present invention provides an expression vectorcomprising the nucleic acid encoding the polypeptide monomer describedabove.

In another aspect, the present invention provides a host celltransfected with the expression vector described above.

In another aspect, the present invention provides a method foridentifying a compound that increases or decreases ion flux through anvoltage-gated potassium channel, the method comprising the steps of: (i)contacting the compound with a eukaryotic host cell or cell membrane inwhich has been expressed a polypeptide monomer comprising an alphasubunit of a potassium channel, the polypeptide monomer: (a) having theability to form, with at least one additional Elk alpha subunit, apotassium channel having the characteristic of voltage gating; (b)having a monomer P-S6 region that has greater than 80% amino acidsequence identity to an hElk P-S6 region; and (c) specifically bindingto polyclonal antibodies generated against SEQ ID NO:1; and (ii)determining the functional effect of the compound upon the cell or cellmembrane expressing the potassium channel.

In one embodiment, the increased or decreased flux of ions is determinedby measuring changes in current or voltage. In another embodiment, thepolypeptide monomer polypeptide is recombinant. In another embodiment,the potassium channel is homomeric.

In another embodiment, the present invention provides a method ofdetecting the presence of hElk in human tissue, the method comprisingthe steps of: (i) isolating a biological sample; (ii) contacting thebiological sample with a hElk-specific reagent that selectivelyassociates with hElk; and, (iii) detecting the level of hElk-specificreagent that selectively associates with the sample.

In one embodiment, the hElk-specific reagent is selected from the groupconsisting of: hElk specific antibodies, hElk specific oligonucleotideprimers, and hElk nucleic acid probes.

In another aspect, the present invention provides, in a computer system,a method of screening for mutations of hElk genes, the method comprisingthe steps of: (i) entering into the computer a first nucleic acidsequence encoding an voltage-gated potassium channel protein having anucleotide sequence of SEQ ID NO:2, and conservatively modified versionsthereof; (ii) comparing the first nucleic acid sequence with a secondnucleic acid sequence having substantial identity to the first nucleicacid sequence; and (iii) identifying nucleotide differences between thefirst and second nucleic acid sequences.

In one embodiment, the second nucleic acid sequence is associated with adisease state.

In another aspect, the present invention provides, in a computer system,a method for identifying a three-dimensional structure of hElkpolypeptides, the method comprising the steps of: (i) entering into thecomputer an amino acid sequence of at least 25, 50 or 100 amino acids ofa potassium channel monomer or at least 75, 150 or 300 nucleotides of anucleic acid encoding the polypeptide, the polypeptide having an aminoacid sequence of SEQ ID NO:1, and conservatively modified versionsthereof; and (ii) generating a three-dimensional structure of thepolypeptide encoded by the amino acid sequence.

In one embodiment, the amino acid sequence is a primary structure andwherein said generating step includes the steps of: (i) forming asecondary structure from said primary structure using energy termsdetermined by the primary structure; and (ii) forming a tertiarystructure from said secondary structure using energy terms determined bysaid secondary structure. In another embodiment, the generating stepincludes the step of forming a quaternary structure from said tertiarystructure using anisotropic terms determined by the tertiary structure.In another embodiment, the methods further comprises the step ofidentifying regions of the three-dimensional structure of the proteinthat bind to ligands and using the regions to identify ligands that bindto the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid alignment of Drosophila (SEQ ID NO:9) and human (SEQID NO:1) Elk. Identical residues are shaded. The numbers at the rightmargin indicate amino acid position. Gaps in alignment are representedby dashes.

FIG. 2. mRNA distribution of human Elk. The height of the bars indicatesrelative expression levels. Data was generated using mRNA dot blots. ThehElk probe used here detects a single band of approximately 4.5 Kb innorthern blot analysis. Tissue names are given below the bars. Highlevels of expression are found in CNS tissues and the pituitary gland.

FIG. 3. Expression of hElk in Xenopus oocytes. (A) hElk currentselicited in response to 480 ms depolarizations from −80 mV to +80 mV in20 mV increments. The holding potential was −90 mV and the tail currentswere measured at −120 mV. Note that there is substantial inactivation athigher voltages. (B) Plot of tail current amplitude vs. holdingpotential for the traces shown in (A). A single boltzman fit of the datayields a half-activation voltage of −34 mV. (C) Blockade of hElk by 3 mMbarium. Currents were elicited by 480 ms depolarizations to +20 mV froma holding potential of −90 mV, with tail currents measured at −120 mV.Traces show control current, block by barium, and reversal of blockfollowing washout of barium.

FIG. 4. Expression of hElk in Chinese Hamster Ovary Cells. (A) Currentselicited from a CHO cell expressing hElk in response to depolarizationto −60, +30, and +60 mV from a holding potential of −80 mV. Tailcurrents were measured at −40 mV. Note the fast-inactivating componentseen at +30 mV and +60 mV. (B) Current/voltage relationship for a CHOcell expressing Elk. There is a substantial reduction in hElk current athigh voltages due to inactivation. (C) Reactivation of hElk afterrecovery from inactivation. A four second pulse to +40 mV was used toactivate hElk. This pulse was followed by a 12.5 ms repolarization to−90 mV to allow for recovery from inactivation and a second depolarizingpulse to reactivate hElk ranging from −2-mV to +90 mV in 10 mVincrements.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides for the first time a nucleic acidencoding human Elk, identified and cloned from human tissue. Thispolypeptide monomer is a member of the “Kv” superfamily of potassiumchannel monomers, the “Eag” (ether a go-go) family of potassium channelmonomer, and the “Elk” subfamily of potassium channel monomers. Membersof this subfamily are polypeptide monomers that are subunits ofvoltage-gated potassium channels having six transmembrane regions(K=potassium, v=voltage-gated). Voltage-gated potassium channels havesignificant roles in maintaining the resting potential and incontrolling excitability of a cell.

The invention also provides methods of screening for activators andinhibitors of voltage-gated potassium channels that contain a hElksubunit. Such modulators of voltage-gated channel activity are usefulfor treating disorders involving abnormal ion flux, e.g., disorders ofthe endocrine system, CNS disorders such as migraines, hearing andvision problems, psychotic disorders, seizures, psychotic disorders, andas neuroprotective agents (e.g., to prevent stroke).

Furthermore, the invention provides assays for hElk activity where hElkacts as a direct or indirect reporter molecule. Such uses of hElk as areporter molecule in assay and detection systems have broadapplications, e.g., hElk can be used as a reporter molecule to measurechanges in potassium concentration, membrane potential, current flow,ion flux, transcription, signal transduction, receptor-ligandinteractions, second messenger concentrations, in vitro, in vivo, and exvivo. In one embodiment, hElk can be used as an indicator of currentflow in a particular direction (e.g., outward or inward potassium flow),and in another embodiment, hElk can be used as an indirect reporter viaattachment to a second reporter molecule such as green fluorescentprotein.

The invention also provides for methods of detecting hElk nucleic acidand protein expression, allowing investigation of the channel diversityprovided by hElk, as well as diagnosis of disease caused by abnormal ionflux, e.g., endocrine system disorders, CNS disorders such as migraines,hearing and vision problems, psychotic disorders, and seizures. Inparticular, hElk has been mapped to chromosome 12 (see Example III).hElk can therefore be used as a marker for diagnosis of diseases linkedto the hElk gene on chromosome 12.

Finally, the invention provides for a method of screening for mutationsof hElk genes or proteins. The invention includes, but is not limitedto, methods of screening for mutations in hElk with the use of acomputer. Similarly, the invention provides for methods of identifyingthe three-dimensional structure of hElk, as well as the resultingcomputer readable images or data that comprise the three dimensionalstructure of hElk. Other methods for screening for mutations of hElkgenes or proteins include high density oligonucleotide arrays, PCR,immunoassays and the like.

Functionally, hElk is an alpha subunit of an voltage-gated potassiumchannel. Typically, such voltage-gated channels are heteromeric orhomomeric and contain four alpha subunits or monomers each with sixtransmembrane domains. Heteromeric Elk channels can comprise one or morehElk alpha subunits along with one or more additional alpha subunitsfrom the Kv superfamily or the Eag family, preferably from the Elksubfamily. hElk channels may also be homomeric. In addition, suchchannels may comprise one or more auxiliary beta subunits. The presenceof hElk in an voltage-gated potassium channel may also modulate theactivity of the heteromeric channel and thus enhance channel diversity.Channel diversity is also enhanced with alternatively spliced forms ofhElk.

Structurally, the nucleotide sequence of human Elk (SEQ ID NO:2) encodesa polypeptide monomer of approximately 1083 amino acids with a predictedmolecular weight of approximately 125 kDa (SEQ ID NO:1) and a predictedrange of 120-130 kDa. In particular, the amino acid sequence of hElk hasa “pore to S6” or “P-S6” region (approximately amino acids 452 to 514,see, e.g., amino acids 452-514 of SEQ ID NO:1, hElk) that has aconserved amino acid sequence. Another conserved amino acid region isthe extended P-S6 region, which includes some cytoplasmic sequence, fromamino acids 452-710. Related Elk genes from other species share at leastabout 80%, preferably 85, 90 or 95% amino acid identity in theseregions.

The present invention also provide polymorphic variants of the hElkdepicted in SEQ ID NO:1: variant #1, in which a valine residue issubstituted for the isoleucine residue at amino acid position 1064;variant #2, in which an isoleucine residue is substituted for theleucine residue at amino acid position 1060; and variant #3, in which aserine residue is substituted for the alanine residue at amino acidposition 744.

Specific regions of hElk nucleotide and amino acid sequence may be usedto identify polymorphic variants, interspecies homologs, and alleles ofhElk. This identification can be made in vitro, e.g., under stringenthybridization conditions and sequencing, or by using the sequenceinformation in a computer system for comparison with other nucleotidesequences, or using antibodies raised to hElk. Typically, identificationof polymorphic variants, orthologs, and alleles of hElk is made bycomparing the amino acid sequence (or the nucleic acid encoding theamino acid sequence) of the P-S6 region (approximately amino acids452-514 of hElk, see SEQ ID NO:1 for example), or by comparing the aminoacid sequence of the extended P-S6 region (amino acids 452-710). Aminoacid identity of approximately at least 80% or above, preferably 85%,most preferably 90-95% or above in the P-S6 region or the extended P-S6region typically demonstrates that a protein is a polymorphic variant,interspecies homolog, or allele of hElk. Sequence comparison can beperformed using any of the sequence comparison algorithms discussedbelow.

Polymorphic variants, interspecies homologs, and alleles of hElk can beconfirmed by expressing or co-expressing the putative hElk polypeptidemonomer and examining whether it forms a potassium channel withElk/Kv/Eag functional characteristics, such as voltage-gating. Thisassay is used to demonstrate that a protein having about 80% or greater,preferably 85%, 90%, or 95% or greater amino acid identity to the“pore-S6” region of hElk shares the same functional characteristics ashElk and is therefore a species of hElk. Typically, hElk having theamino acid sequence of SEQ ID NO:1 is used as a positive control incomparison to the putative hElk protein to demonstrate theidentification of a polymorphic variant, ortholog, or allele of hElk.

hElk nucleotide and amino acid sequence information may also be used toconstruct models of voltage-gated potassium channels in a computersystem. These models are subsequently used to identify compounds thatcan activate or inhibit voltage-gated potassium channels comprisinghElk. Such compounds that modulate the activity of channels comprisinghElk can be used to investigate the role of hElk in modulation ofchannel activity and in channel diversity.

The isolation of biologically active hElk for the first time provides ameans for assaying for inhibitors and activators of voltage-gatedpotassium channels that comprise hElk subunits. Biologically active hElkis useful for testing inhibitors and activators of voltage-gatedpotassium channels comprising subunits of hElk and other Kv membersusing in vivo and in vitro expression that measure, e.g., changes involtage or current. Such activators and inhibitors identified using anvoltage-gated potassium channel comprising at least one hElk subunit,preferably four Elk subunits, can be used to further study voltagegating, channel kinetics and conductance properties of potassiumchannels. Such activators and inhibitors are useful as pharmaceuticalagents for treating diseases involving abnormal ion flux, e.g.,endocrine and CNS disorders, as described above. Methods of detectinghElk and expression of channels comprising hElk are also useful fordiagnostic applications for diseases involving abnormal ion flux, e.g.,CNS disorders, endocrine disorders, and other disorders as describedabove. For example, chromosome localization of the gene encoding humanElk can be used to identify diseases caused by and associated with hElk.Methods of detecting hElk are also useful for examining the role of hElkin channel diversity and modulation of channel activity.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The phrase “voltage-gated” activity or “voltage-gating” refers to acharacteristic of a potassium channel composed of individual polypeptidemonomers or subunits. Generally, the probability of a voltage-gatedpotassium channel opening increases as a cell is depolarized.Voltage-gated potassium channels primarily allow efflux of potassiumbecause they have greater probabilities of being open at membranepotentials more positive than the membrane potential for potassium(E_(K)) in typical cells. E_(K), or the membrane potential forpotassium, depends on the relative concentrations of potassium foundinside and outside the cell membrane, and is typically between −60 and−100 mV for mammalian cells. E_(K) is the membrane potential at whichthere is no net flow of potassium ion because the electrical potential(i.e., voltage potential) driving potassium influx is balanced by theconcentration gradient (the [K⁺] potential) directing potassium efflux.This value is also known as the “reversal potential” or the “Nernst”potential for potassium. Some voltage-gated potassium channels undergoinactivation, which can reduce potassium efflux at higher membranepotentials. Potassium channels can also allow potassium influx incertain instances when they remain open at membrane potentials negativeto E_(K)(see, e.g., Adams & Normer, in Potassium Channels, pp. 40-60(Cook, ed., 1990)). The characteristic of voltage gating can be measuredby a variety of techniques for measuring changes in current flow and ionflux through a channel, e.g., by changing the [K⁺] of the externalsolution and measuring the activation potential of the channel current(see, e.g., U.S. Pat. No. 5,670,335), by measuring current with patchclamp techniques or voltage clamp under different conditions, and bymeasuring ion flux with radiolabeled tracers or voltage-sensitive dyesunder different conditions.

“Homomeric channel” refers to an hElk channel composed of identicalalpha subunits, whereas “heteromeric channel” refers to an hElk channelcomposed of at least one hElk alpha subunit plus at least one otherdifferent type of alpha subunit from a related gene family such as theKv superfamily or the Eag family, preferably from the Elk subfamily.Both homomeric and heteromeric channels can include auxiliary betasubunits. Typically, the channel is composed of four alpha subunits andthe channel can be heteromeric or homomeric.

A “beta subunit” is a polypeptide monomer that is an auxiliary subunitof a potassium channel composed of alpha subunits; however, betasubunits alone cannot form a channel (see, e.g., U.S. Pat. No.5,776,734). Beta subunits are known, for example, to increase the numberof channels by helping the alpha subunits reach the cell surface, changeactivation kinetics, and change the sensitivity of natural ligandsbinding to the channels. Beta subunits can be outside of the pore regionand associated with alpha subunits comprising the pore region. They canalso contribute to the external mouth of the pore region.

The phrase “P-S6 region” (pore to S6) refers to the region of hElk thatstructurally identifies this particular protein (approximately aminoacids 452-514 of hElk, see SEQ ID NO:1). The phrase “extended P-S6region” refers to another region of hElk that can also be used tostructurally identify this protein (approximately amino acids 452-710 ofhElk, see SEQ ID NO:1). These regions can be used to identify hElkpolymorphic variants, orthologs, and hElk alleles of hElk, through aminoacid sequence identity comparison using a sequence comparison algorithmsuch as BLASTP or PILEUP. P-S6 comprises the pore region arid the S6transmembrane region, while the extended P-S6 region includescytoplasmic sequences. (see, e.g., Ackerman & Clapham, New Engl. J. Med.336:1575-1586 (1997); Jan & Jan, Annu. Rev. Neurosci. 20:91-123 (1997)).

“hElk” refers to a polypeptide that is a subunit or monomer of anvoltage-gated potassium channel, a member of the Elk subfamily, a memberof the Eag family, and a member of the Kv superfamily of potassiumchannel monomers. When hElk is part of a potassium channel, either ahomomeric or heteromeric potassium channel, the channel hasvoltage-gated activity. The term hElk therefore refers to polymorphicvariants, alleles, mutants, and interspecies homologs that: (1) have aP-S6 region or an extended P-S6 region that has greater than about 80%amino acid sequence identity, preferably about 85, 90, or 95% amino acidsequence identity, to a hElk P-S6 region or an extended P-S6 region; (2)bind to antibodies raised against an immunogen comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1, amino acids452-514 of SEQ ID NO:1, amino acids 452-710 of SEQ ID NO:1 andconservatively modified variants thereof; (3) specifically hybridizeunder stringent hybridization conditions to a sequence selected from thegroup consisting of SEQ ID NO:2, the nucleotide sequence encoding aminoacids 452-514 of SEQ ID NO:1, the nucleotide sequence encoding aminoacids 452-710 of SEQ ID NO:1, and conservatively modified variantsthereof; or (4) are amplified by primers that specifically hybridizeunder stringent hybridization conditions to the same sequence as aprimer set consisting of SEQ ID NO:3 and SEQ ID NO:4 or SEQ ID NO:5 andSEQ ID NO:6.

The phrase “functional effects” in the context of assays for testingcompounds affecting a channel comprising hElk includes the determinationof any parameter that is indirectly or directly under the influence ofthe channel. It includes changes in ion flux and membrane potential, andalso includes other physiologic effects such as increases or decreasesof transcription or hormone release.

“Determining the functional effect” refers to examining the effect of acompound that increases or decreases ion flux on a cell or cell membranein terms of cell and cell membrane function. The ion flux can be any ionthat passes through a channel and analogues thereof, e.g., potassium,rubidium, sodium. Preferably, the term refers to the functional effectof the compound on the channels comprising hElk, e.g., changes in ionflux including radioisotopes, current amplitude, membrane potential,current flow, transcription, protein binding, phosphorylation,dephosphorylation, second messenger concentrations (cAMP, cGMP, Ca²⁺,IP₃) and other physiological effects such as hormone andneurotransmitter release, as well as changes in voltage and current.Such functional effects can be measured by any means known to thoseskilled in the art, e.g., patch clamping, voltage-sensitive dyes, wholecell currents, radioisotope efflux, inducible markers, and the like.

“Inhibitors,” “activators” or “modulators” of voltage-gated potassiumchannels comprising hElk refer to inhibitory or activating moleculesidentified using in vitro and in vivo assays for hElk channel function.Inhibitors are compounds that decrease, block, prevent, delayactivation, inactivate, desensitize, or down regulate the channel.Activators are compounds that increase, open, activate, facilitate,enhance activation, sensitize or up regulate channel activity. Suchassays for inhibitors and activators include e.g., expressing hElk incells or cell membranes and then measuring flux of ions through thechannel and determining changes in polarization (i.e., electricalpotential). Alternatively, cells expressing endogenous hElk channels canbe used in such assays. To examine the extent of inhibition, samples orassays comprising an hElk channel are treated with a potential activatoror inhibitor and are compared to control samples without the inhibitor.Control samples (untreated with inhibitors) are assigned a relative hElkactivity value of 100%. Inhibition of channels comprising hElk isachieved when the hElk activity value relative to the control is about90%, preferably 50%, more preferably 25-0%. Activation of channelscomprising hElk is achieved when the hElk activity value relative to thecontrol is 110%, more preferably 150%, most preferably at least 200-500%higher or 1000% or higher.

“Biologically active” hElk refers to hElk that has the ability to form apotassium channel having the characteristic of voltage-gating tested asdescribed above.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. Inparticular, an isolated hElk nucleic acid is separated from open readingframes that flank the hElk gene and encode proteins other than hElk. Theterm “purified” denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Particularly, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition. An example ofpotassium channel splice variants is discussed in Leicher, et al., J.Biol. Chem. 273 (52):35095-35101 (1998).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)    (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 50 to 350 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins for which antisera or monoclonal antibodies are available(e.g., the polypeptide of SEQ ID NO:1 can be made detectable, e.g., byincorporating a radiolabel into the peptide, and used to detectantibodies specifically reactive with the peptide).

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 80% identity, preferably 85%, 90%, or 95% identity over aspecified region such as the hElk P-S6 region or the extended P-S6region), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the compliment of a testsequence. Preferably, the identity exists over a region that is at leastabout 25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For high stringency hybridization, a positivesignal is at least two times background, preferably 10 times backgroundhybridization. Exemplary high stringency or stringent hybridizationconditions include: 50% formamide, 5×SSC and 1% SDS incubated at 42° C.or 5×SSC and 1% SDS incubated at 65° C., with a wash in 0.2×SSC and 0.1%SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cased, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc. (1985)). Techniques for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992));

An “anti-hElk” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the hElk gene, cDNA, or asubsequence thereof.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to hElk, encoded in SEQ ID NO:1, splice variants, or portionsthereof, can be selected to obtain only those polyclonal antibodies thatare specifically immunoreactive with hElk and not with other proteins,except for polymorphic variants, orthologs, and alleles of hElk. Thisselection may be achieved by subtracting out antibodies that cross-reactwith molecules such as Drosophila Elk and other Elk orthologs, and withother family members such as rat and mouse Eag. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select antibodies specifically immunoreactive witha protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity). Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, e.g., cultured cells, explants, and cells in vivo.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains hElk or nucleic acid encoding hElk protein. Suchsamples include, but are not limited to, tissue isolated from humans.Biological samples may also include sections of tissues such as frozensections taken for histologic purposes. A biological sample is typicallyobtained from a eukaryotic organism, preferably eukaryotes such asfungi, plants, insects, protozoa, birds, fish, reptiles, and preferablya mammal such as rat, mice, cow, dog, guinea pig, or rabbit, and mostpreferably a primate such as chimpanzees or humans.

III. Isolating the Gene Encoding hELK

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodinghElk

In general, the nucleic acid sequences encoding hElk and related nucleicacid sequence homologs are cloned from cDNA and genomic DNA libraries orisolated using amplification techniques with oligonucleotide primers.For example, hElk sequences are typically isolated from human nucleicacid (genomic or cDNA) libraries by hybridizing with a nucleic acidprobe or polynucleotide, the sequence of which can be derived from SEQID NO:2, preferably from the region encoding the P-S6 region or from theregion encoding the extended P-S6 region. A suitable tissue from whichElk RNA and cDNA can be isolated is brain tissue such as whole brain orhippocampus (see, e.g., FIG. 2).

Amplification techniques using primers can also be used to amplify andisolate hElk from DNA or RNA. The following primers can also be used toamplify a sequence of hElk: ATGCCGGCCATGCGGGGCCTCCT (SEQ ID NO:3),AGATGGCAGCACACCTGGCAACGCTG (SEQ ID NO:4) and GCCCATCTGCTGAAGACGGTGCGC(SEQ ID NO:5), CGAAGCCCACGCTGGTGAGGCTGCTG (SEQ ID NO:6). These primerscan be used, e.g., to amplify either the full length sequence or a probeof one to several hundred nucleotides, which is then used to screen ahuman library for full-length hElk.

Nucleic acids encoding hElk can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using the sequence of SEQ ID NO:1.

Human Elk polymorphic variants, orthologs, and alleles that aresubstantially identical to the P-S6 region or the extended P-S6 regionof hElk can be isolated using hElk nucleic acid probes andoligonucleotides under stringent hybridization conditions, by screeninglibraries. Alternatively, expression libraries can be used to clone hElkand hElk polymorphic variants, orthologs, and alleles by detectingexpressed homologs immunologically with antisera or purified antibodiesmade against hElk or portions thereof (e.g., the P-S6 region or theextended P-S6 region of hElk), which also recognize and selectively bindto the hElk homolog.

To make a cDNA library, one should choose a source that is rich in hElkmRNA, e.g., tissue such as whole brain or hippocampus (see, e.g., FIG.2). The mRNA is then made into cDNA using reverse transcriptase, ligatedinto a recombinant vector, and transfected into a recombinant host forpropagation, screening and cloning. Methods for making and screeningcDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating hElk nucleic acid and its homologscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of hElkdirectly from mRNA, from cDNA, from genomic libraries or cDNA libraries.Degenerate oligonucleotides can be designed to amplify hElk homologsusing the sequences provided herein. Restriction endonuclease sites canbe incorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of hElkencoding mRNA in physiological samples, for nucleic acid sequencing, orfor other purposes. Genes amplified by the PCR reaction can be purifiedfrom agarose gels and cloned into an appropriate vector.

Gene expression of hElk can also be analyzed by techniques known in theart, e.g., reverse transcription and amplification of mRNA, isolation oftotal RNA or poly A⁺ RNA, northern blotting, dot blotting, in situhybridization, RNase protection, high density polynucleotide arraytechnology and the like.

Synthetic oligonucleotides can be used to construct recombinant hElkgenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the hElk gene. The specificsubsequence is then ligated into an expression vector.

The gene for hElk is typically cloned into intermediate vectors beforetransformation into prokaryotic or eukaryotic cells for replicationand/or expression. These intermediate vectors are typically prokaryotevectors, e.g., plasmids, or shuttle vectors.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding hElk, one typically subclones hElk into an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al. and Ausubel et al, supra. Bacterial expressionsystems for expressing the hElk protein are available in, e.g., E. coli,Bacillus sp., and Salmonella (Palva et al. Gene 22:229-235 (1983);Mosbach et al., Nature 302:543-545 (1983). Kits for such expressionsystems are commercially available. Eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available.

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the hElk encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding hElkand signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. Additional elementsof the cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to tansport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, bactilovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the CMV promoter, SV40early promoter, SV40 later promoter, metallothionein promoter, murinemammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrinpromoter, or other promoters shown effective for expression ineukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with ahElk encoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically-included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of hElkprotein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressinghElk.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofhElk, which is recovered from the culture using standard techniquesidentified below.

IV. Purification of hELK Polypeptides

Either naturally occurring or recombinant hElk can be purified for usein functional assays. Naturally occurring hElk monomers can be purified,e.g., from human tissue such as whole brain or hippocampus, and anyother source of a hElk homolog. Recombinant hElk monomers can bepurified from any suitable expression system.

The hElk monomers may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant hElk monomersare being purified. For example, proteins having established molecularadhesion properties can be reversible fused to the hElk monomers. Withthe appropriate ligand, the hElk monomers can be selectively adsorbed toa purification column and then freed from the column in a relativelypure form. The fused protein is then removed by enzymatic activity.Finally the hElk monomers could be purified using immunoaffinitycolumns.

A. Purification of hElk Monomers from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of the hElkmonomers inclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. Human Elk monomers areseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni—NTA agarose resin.

Alternatively, it is possible to purify the hElk monomers from bacteriaperiplasm. After lysis of the bacteria, when the hElk monomers areexported into the periplasm of the bacteria, the periplasmic fraction ofthe bacteria can be isolated by cold osmotic shock in addition to othermethods known to skill in the art. To isolate recombinant proteins fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

B. Standard Protein Separation Techniques for Purifying the hELKMonomers

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of the hElk monomers can be used to isolated itfrom proteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

The hElk monomers can also be separated from other proteins on the basisof its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

V. Immunological Detection of hELK

In addition to the detection of hElk genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect the hElk monomers. Immunoassays can be used to qualitatively orquantitatively analyze the hElk monomers. A general overview of theapplicable technology can be found in Harlow & Lane, Antibodies: ALaboratory Manual (1988).

A. Antibodies to hElk Monomers

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with the hElk monomers are known to those of skill in theart (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2d ed. 1986); and Kohler & Milstein, Nature 256:495497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of immunogens comprising portions of hElk monomers may be usedto produce antibodies specifically reactive with hElk monomers. Forexample, recombinant hElk monomers or an antigenic fragment thereof,such as the P-S6 region or the extended P-S6 region, can be isolated asdescribed herein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-hElkproteins, other Elk orthologs, other Eag family members, or other Kvsuperfamily members, using a competitive binding immunoassay. Specificpolyclonal antisera and monoclonal antibodies will usually bind with aK_(d) of at least about 0.1 mM, more usually at least about 1 μM,preferably at least about 0.1 μM or better, and most preferably, 0.01 μMor better.

Once the specific antibodies against a hElk are available, the hElk canbe detected by a variety of immunoassay methods. For a review ofimmunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

B. Immunological Binding Assays

The hElk can be detected and/or quantified using any of a number of wellrecognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7^(th) ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case the hElk or an antigenicsubsequence thereof). The antibody (e.g., anti-hElk) may be produced byany of a number of means well known to those of skill in the art and asdescribed above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled hElk polypeptide or alabeled anti-hElk antibody. Alternatively, the labeling agent may be athird moiety, such a secondary antibody, which specifically binds to theantibody/hElk complex (a secondary antibody is typically specific toantibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol. 111: 1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting the hElk in samples may be either competitiveor noncompetitive. Noncompetitive immunoassays are assays in which theamount of antigen is directly measured. In one preferred “sandwich”assay, for example, the anti-hElk subunit antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture hElk present in the test sample. ThehElk monomers are thus immobilized and then bound by a labeling agent,such as a second hElk antibody bearing a label. Alternatively, thesecond antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety.

Competitive Assay Formats

In competitive assays, the amount of the hElk present in the sample ismeasured indirectly by measuring the amount of known, added (exogenous)hElk displaced (competed away) from an anti-hElk antibody by the unknownhElk present in a sample. In one competitive assay, a known amount ofthe hElk is added to a sample and the sample is then contacted with anantibody that specifically binds to the hElk. The amount of exogenoushElk bound to the antibody is inversely proportional to theconcentration of the hElk present in the sample. In a particularlypreferred embodiment, the antibody is immobilized on a solid substrate.The amount of hElk bound to the antibody may be determined either bymeasuring the amount of hElk present in a hElk/antibody complex, oralternatively by measuring the amount of remaining uncomplexed protein.The amount of hElk may be detected by providing a labeled hElk molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known hElk is immobilized on a solid substrate. A knownamount of anti-hElk antibody is added to the sample, and the sample isthen contacted with the immobilized hElk. The amount of anti-hElkantibody bound to the known immobilized hElk is inversely proportionalto the amount of hElk present in the sample. Again, the amount ofimmobilized antibody may be detected by detecting either the immobilizedfraction of antibody or the fraction of the antibody that remains insolution. Detection may be direct where the antibody is labeled orindirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations for hElk. For example, a protein at leastpartially encoded by SEQ ID NO:2 or an immunogenic region thereof, suchas the P-S6 region (amino acids 452-514), or an immunogenic regionthereof, such as the extended P-S6 region (amino acids 452-710), can beimmobilized to a solid support. Other proteins such as other Elksubfamily members, e.g., Drosophila Elk, are added to the assay so as tocompete for binding of the antisera to the immobilized antigen. Theability of the added proteins to compete for binding of the antisera tothe immobilized protein is compared to the ability of the hElk encodedby SEQ ID NO:1 to compete with itself. The percent crossreactivity forthe above proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with each of the addedproteins listed above are selected and pooled. The cross-reactingantibodies are optionally removed from the pooled antisera byimmunoabsorption with the added considered proteins, e.g., distantlyrelated homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele, ortholog, or polymorphic variant ofhElk, to the immunogen protein. In order to make this comparison, thetwo proteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required to inhibit 50% of binding is less than 10 timesthe amount of the protein encoded by hElk that is required to inhibit50% of binding, then the second protein is said to specifically bind tothe polyclonal antibodies generated to the respective hElk immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of the hElk in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind hElk. The anti-hElk antibodies specifically bindto hElk on the solid support. These antibodies may be directly labeledor alternatively may be subsequently detected using labeled antibodies(e.g., labeled sheep anti-mouse antibodies) that specifically bind tothe anti-hElk antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize hElk, orsecondary antibodies that recognize anti-hElk antibodies.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of hELK

A. Assays

Human Elk monomers and hElk alleles, orthologs, and polymorphic variantsare subunits of voltage-gated potassium channels. The activity of apotassium channel comprising hElk can be assessed using a variety of invitro and in vivo assays, e.g., measuring current, measuring membranepotential, measuring ion flux, e.g., potassium or rubidium, measuringpotassium concentration, measuring second messengers and transcriptionlevels, using potassium-dependent yeast growth assays, and using e.g.,voltage-sensitive dyes, radioactive tracers, and patch-clampelectrophysiology.

Furthermore, such assays can be used to test for inhibitors andactivators of channels comprising hElk. Such modulators of a potassiumchannel are useful for treating various disorders involving potassiumchannels. Treatment of dysfunctions include, e.g., endocrine disorders,CNS disorders such as migraines, hearing and vision problems, psychoticdisorders, seizures, and use as neuroprotective agents (e.g., to preventstroke). Such modulators are also useful for investigation of thechannel diversity provided by hElk and the regulation/modulation ofpotassium channel activity provided by hElk.

Modulators of the potassium channels are tested using biologicallyactive hElk, either recombinant or naturally occurring. Human Elk can beisolated, co-expressed or expressed in a cell, or expressed in amembrane derived from a cell. In such assays, hElk is expressed alone toform a homomeric potassium channel or is co-expressed with a secondalpha subunit (e.g., another Kv superfamily member or an Eag familymember, preferably an Elk subfamily member) so as to form a heteromericpotassium channel. Elk can also be expressed with additional betasubunits. Modulation is tested using one of the in vitro or in vivoassays described above. Samples or assays that are treated with apotential potassium channel inhibitor or activator are compared tocontrol samples without the test compound, to examine the extent ofmodulation. Control samples (untreated with activators or inhibitors)are assigned a relative potassium channel activity value of 100.Inhibition of channels comprising hElk is achieved when the potassiumchannel activity value relative to the control is about 90%, preferably50%, more preferably 25%. Activation of channels comprising hElk isachieved when the potassium channel activity value relative to thecontrol is 110%, more preferably 150%, more preferable 200% higher.Compounds that increase the flux of ions will cause a detectableincrease in the ion current density by increasing the probability of achannel comprising hElk being open, by decreasing the probability of itbeing closed, by increasing conductance through the channel, and/or byallowing the passage of ions.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing the potassium channel comprising hElk. A preferred means todetermine changes in cellular polarization is by measuring changes incurrent (thereby measuring changes in polarization) with voltage-clampand patch-clamp techniques, e.g., the “cell-attached” mode, the“inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman etal., New Engl. J. Med. 336:1575-1595 (1997)). Whole cell currents areconveniently determined using the standard methodology (see, e.g., Hamilet al., PFlugers. Archiv. 391:85 (1981). Other known assays include:radiolabeled rubidium flux assays and fluorescence assays usingvoltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J.Membrane Biol. 88:67-75 (1988); Daniel et al., J. Pharmacol. Meth.25:185-193 (1991); Holevinsky et al., J. Membrane Biology 137:59-70(1994)). Assays for compounds capable of inhibiting or increasingpotassium flux through the channel proteins comprising hElk can beperformed by application of the compounds to a bath solution in contactwith and comprising cells having a channel of the present invention(see, e.g., Blatz et al., Nature 323:718-720 (1986); Park, J. Physiol.481:555-570 (1994)). Generally, the compounds to be tested are presentin the range from 1 pM to 100 mM.

The effects of the test compounds upon the function of the channels canbe measured by changes in the electrical currents or ionic flux or bythe consequences of changes in currents and flux. Changes in electricalcurrent or ionic flux are measured by either increases or decreases influx of ions such as potassium or rubidium ions. The cations can bemeasured in a variety of standard ways. They can be measured directly byconcentration changes of the ions or indirectly by membrane potential orby radio-labeling of the ions. Consequences of the test compound on ionflux can be quite varied. Accordingly, any suitable physiological changecan be used to assess the influence of a test compound on the channelsof this invention. The effects of a test compound can be measured by atoxin binding assay. When the functional consequences are determinedusing intact cells or animals, one can also measure a variety of effectssuch as transmitter release (e.g., dopamine), hormone release (e.g.,insulin), transcriptional changes to both known and uncharacterizedgenetic markers (e.g., northern blots), cell volume changes (e.g., inred blood cells), immunoresponses (e.g., T cell activation), changes incell metabolism such as cell growth or pH changes, and changes inintracellular second messengers such as Ca²⁺, or cyclic nucleotides.

Preferably, the Elk that is a part of the potassium channel used in theassay will have the sequence displayed in SEQ ID NO:1 or aconservatively modified variant thereof. Alternatively, the Elk of theassay will be derived from a eukaryote and include an amino acidsubsequence having substantial amino acid sequence identity to the P-S6region or the extended P-S6 region of hElk. Generally, the amino acidsequence identity will be at least 80%, preferably at least 85 to 90%,most preferably at least 95%.

Human Elk orthologs will generally confer substantially similarproperties on a channel comprising such hElk, as described above. In apreferred embodiment, the cell placed in contact with a compound that issuspected to be a hElk homolog is assayed for increasing or decreasingion flux in a eukaryotic cell, e.g., an oocyte of Xenopus (e.g., Xenopuslaevis) or a mammalian cell such as a CHO or HeLa cell. Channels thatare affected by compounds in ways similar to hElk are consideredhomologs or orthologs of hElk.

B. Modulators

The compounds tested as modulators of Elk channels comprising a humanElk subunit can be any small chemical compound, or a biological entity,such as a protein, sugar, nucleic acid or lipid. Alternatively,modulators can be genetically altered versions of a human Elk subunit.Typically, test compounds will be small chemical molecules and peptides.Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g. U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see,e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.5,593,853), small organic molecule libraries (see, e.g.,benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S.Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S.Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressinga Elk channel comprising a human Elk subunit is attached to a solidphase substrate. In the high throughput assays of the invention, it ispossible to screen up to several thousand different modulators orligands in a single day. In particular, each well of a microtiter platecan be used to run a separate assay against a selected potentialmodulator, or, if concentration or incubation time effects are to beobserved, every 5-10 wells can test a single modulator. Thus, a singlestandard microtiter plate can assay about 96 modulators. If 1536 wellplates are used, then a single plate can easily assay from about 100about 1500 different compounds. It is possible to assay severaldifferent plates per day; assay screens for up to about 6,000-20,000different compounds is possible using the integrated systems of theinvention.

VII. Computer Assisted Drug Design Using hELK

Yet another assay for compounds that modulate the activities of hElkinvolves computer assisted drug design, in which a computer system isused to generate a three-dimensional structure of hElk based on thestructural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apre-established algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands or otherpotassium channel subunits. These regions are then used to identifyligands that bind to the protein or region where hElk interacts withother potassium channel subunits.

The three-dimensional structural model of the protein is generated byentering channel protein amino acid sequences of at least 25, 50, 75 or100 amino acid residues or corresponding nucleic acid sequences encodingan hElk monomer into the computer system. The amino acid sequence ofeach of the monomers is selected from the group consisting of SEQ IDNO:1 and a conservatively modified versions thereof. The amino acidsequence represents the primary sequence or subsequence of each of theproteins, which encodes the structural information of the protein. Atleast 25, 50, 75, or 100 residues of the amino acid sequence (or anucleotide sequence encoding at least about 25, 50, 75 or 100 aminoacids) are entered into the computer system from computer keyboards,computer readable substrates that include, but are not limited to,electronic storage media (e.g., magnetic diskettes, tapes, cartridges,and chips), optical media (e.g., CD ROM), information distributed byinternet sites, and by RAM. The three-dimensional structural model ofthe channel protein is then generated by the interaction of the aminoacid sequence and the computer system, using software known to those ofskill in the art. The resulting three-dimensional computer model canthen be saved on a computer readable substrate.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the monomer and the heteromeric potassium channel proteincomprising four monomers. The software looks at certain parametersencoded by the primary sequence to generate the structural model. Theseparameters are referred to as “energy terms,” or anisotropic terms andprimarily include electrostatic potentials, hydrophobic potentials,solvent accessible surfaces, and hydrogen bonding. Secondary energyterms include van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of hElk protein to identify ligands that bind to hElk. Bindingaffinity between the protein and ligands is determined using energyterms to determine which ligands have an enhanced probability of bindingto the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of hElk genes. Suchmutations can be associated with disease states. Once the variants areidentified, diagnostic assays can be used to identify patients havingsuch mutated genes associated with disease states. Identification of themutated hElk genes involves receiving input of a first nucleic acid,e.g., SEQ ID NO:2, or an amino acid sequence encoding hElk, selectedfrom the group consisting of SEQ ID NO:1, and a conservatively modifiedversions thereof. The sequence is entered into the computer system asdescribed above. The first nucleic acid or amino acid sequence is thencompared to a second nucleic acid or amino acid sequence that hassubstantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in hElk genes, and mutations associatedwith disease states. The first and second sequences described above canbe saved on a computer readable substrate.

Human Elk monomers and the potassium channels containing these hElkmonomers can be used with high density oligonucleotide array technology(e.g., GeneChip™) to identify homologs and polymorphic variants of hElkin this invention. In the case where the homologs being identified arelinked to a known disease, they can be used with GeneChip™ as adiagnostic tool in detecting the disease in a biological sample, see,e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14: 869-876 (1998);Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al., Anal. Biochem.224:110-106 (1995); Lockhart et al., Nat. Biotechnol. 14:1675-1680(1996); Gingeras et al., Genome Res. 8:435-448 (1998); Hacia et al.,Nucleic Acids Res. 26:3865-3866 (1998).

VIII. Cellular Transfection and Gene Therapy

The present invention provides the nucleic acids of hElk for thetransfection of cells in vitro and in vivo. These nucleic acids can beinserted into any of a number of well-known vectors for the transfectionof target cells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The nucleic acid for hElk, under thecontrol of a promoter, then expresses a hElk monomer of the presentinvention, thereby mitigating the effects of absent, partialinactivation, or abnormal expression of the hElk gene.

Such gene therapy procedures have been used to correct acquired andinherited genetic defects, cancer, and viral infection in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies(for a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6 (10):1149-1154 (1998); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51 (1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology (Doerfler & Böhm eds., 1995); and Yu etal., Gene Therapy 1:13-26 (1994)).

Delivery of the gene or genetic material into the cell is the firstcritical step in gene therapy treatment of disease. A large number ofdelivery methods are well known to those of skill in the art.Preferably, the nucleic acids are administered for in vivo or ex vivogene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell.

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described in,e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355 and lipofectionreagents are sold commercially (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids that are suitable for efficientreceptor-recognition lipofection of polynucleotides include those ofFelgner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivoadministration) or target tissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of nucleic acids could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV),and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression of the nucleic acid ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994)). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

pLASN and MFG-S are examples are retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., Proc. Natl. Acad. Sci.U.S.A. 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeuticvector used in a gene therapy trial. (Blaese et al., Science 270:475-480(1995)). Transduction efficiencies of 50% or greater have been observedfor MFG-S packaged vectors (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997)).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used transient expression gene therapy, because they canbe produced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 241:5-10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18(1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al.,Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089(1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors used in gene therapy typically only possess ITRsequences from the AAV genome which are required for packaging andintegration into the host genome. Viral DNA is packaged in a cell line,which contains a helper plasmid encoding the other AAV genes, namely repand cap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. For example, Han et al., Proc. Natl. Acad. Sci. U.S.A.92:9747-9751 (1995), reported that Moloney murine leukemia virus can bemodified to express human heregulin fused to gp70, and the recombinantvirus infects certain human breast cancer cells expressing humanepidermal growth factor receptor. This principle can be extended toother pairs of virus expressing a ligand fusion protein and target cellexpressing a receptor. For example, filamentous phage can be engineeredto display antibody fragments (e.g., FAB or Fv) having specific bindingaffinity for virtually any chosen cellular receptor. Although the abovedescription applies primarily to viral vectors, the same principles canbe applied to nonviral vectors. Such vectors can be engineered tocontain specific uptake sequences thought to favor uptake by specifictarget cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a nucleicacid (gene or cDNA), and re-infused back into the subject organism(e.g., patient). Various cell types suitable for ex vivo transfectionare well known to those of skill in the art (see, e.g., Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) andthe references cited therein for a discussion of how to isolate andculture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+(panb cells), GR-1 (granulocytes),and Iad (differentiated antigen presenting cells) (see Inaba et al., J.Exp. Med. 176:1693-1702 (1992)).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be also administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The nucleicacids are administered in any suitable manner, preferably withpharmaceutically acceptable carriers. Suitable methods of administeringsuch nucleic acids are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

IX. Pharmaceutical Compositions

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered (e.g., nucleic acid, protein,modulatory compounds or transduced cell), as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed., 1989).

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the packaged nucleic acid with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the compound of choice with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant expression of the Elk channels comprising a human Elk alphasubunit, the physician evaluates circulating plasma levels of thevector, vector toxicities, progression of the disease, and theproduction of anti-vector antibodies. In general, the dose equivalent ofa naked nucleic acid from a vector is from about 1 μg to 100 μg for atypical 70 kilogram patient, and doses of vectors which include aretroviral particle are calculated to yield an equivalent amount oftherapeutic nucleic acid.

For administration, compounds and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

Transduced cells are prepared for reinfusion according to establishedmethods (see, e.g., Abrahainsen et al., J. Clin. Apheresis 6:48—53(1991); Carter et al., J. Clin. Apheresis 4:113-117 (1998); Aebersold etal., J. Immunol. Meth. 112:1-7 (1998); Muul et al., J. Immunol. Methods101:171-181 (1987); and Carter et al., Transfusion 27:362-365 (1987)).

X. Kits

Human Elk and its homologs are useful tools for examining expression andregulation of potassium channels. Human Elk-specific reagents thatspecifically hybridize to hElk nucleic acid, such as hElk probes andprimers, and hElk-specific reagents that specifically bind to the hElkprotein, e.g., hElk antibodies are used to examine expression andregulation.

Nucleic acid assays for the presence of hElk DNA and RNA in a sampleinclude numerous techniques are known to those skilled in the art, suchas Southern analysis, northern analysis, dot blots, RNase protection, S1analysis, amplification techniques such as PCR and LCR, and in situhybridization. In in situ hybridization, for example, the target nucleicacid is liberated from its cellular surroundings in such as to beavailable for hybridization within the cell while preserving thecellular morphology for subsequent interpretation and analysis. Thefollowing articles provide an overview of the art of in situhybridization: Singer et al., Biotechniques 4:230-250 (1986); Haase etal., Methods in Virology, vol. VII, pp. 189-226 (1984); and Nucleic AcidHybridization: A Practical Approach (Hames et al., eds. 1987). Inaddition, hElk protein can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing recombinant hElkmonomers) and a negative control.

The present invention also provides for kits for screening modulators ofthe heteromeric potassium channels. Such kits can be prepared fromreadily available materials and reagents. For example, such kits cancomprise any one or more of the following materials: hElk monomers,reaction tubes, and instructions for testing the activities of potassiumchannels containing hElk. A wide variety of kits and components can beprepared according to the present invention, depending upon the intendeduser of the kit and the particular needs of the user. For example, thekit can be tailored for in vitro or in vivo assays for measuring theactivity of a potassium channel comprising a hElk monomer.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following example is provided by way of illustration only and not byway of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I Cloning and Expression of hELK

Using PCR and primers, according to standard conditions, hElk isamplified from brain tissue cDNA, e.g., whole brain or hippocampus cDNA.The following primers were used for amplification:ATGCCGGCCATGCGGGGCCTCCT (SEQ ID NO:3), AGATGGCAGCACACCTGGCAACGCTG (SEQID NO:4).

The cDNA is prepared from total RNA isolated from brain tissue, e.g.,whole brain or hippocampus, according to standard methods. hElk isamplified with the primers described above using the followingconditions: 15 seconds at 96° C., 15 seconds at 72-60° C., and 3 minutesat 72° C. for 40 cycles.

The PCR products are subcloned into plasmids and sequenced according tostandard techniques. The nucleotide and amino acid sequences of hElk areprovided, respectively, in SEQ ID NO:2 and SEQ ID NO:1 (see FIG. 1 foran amino acid alignment of human and Drosophila Elk).

mRNA distribution of hElk was examined according to standard techniques(see FIG. 2).

Example II Expression and Voltage-Gated Activity of Homomeric ChannelsContaining hElk Monomers

hElk monomer was expressed in both Xenopus oocytes and CHO cellsaccording to standard methodology, to demonstrate its ability to formhomomeric potassium channels with voltage-gated activity (see FIGS. 3and 4). Changes in current magnitude can be indirectly measured using areporter voltage-sensitive fluorescent dye (see, e.g., Etts et al.,Chemistry and Physiology of Lipids, 69:137 (1994)). Changes in currentmagnitude can also be measured directly using electrophysiology, and bymeasuring ion flux.

hElk channels are potassium selective and voltage-gated. These channelsopen in a time-dependent manner in response to depolarization, and havea significant probability of opening at voltages more positive thanabout −70 mV. There is substantial reduction in hElk current at highvoltages due to inactivation (see FIG. 4).

Example III Genomic Localization of the hElk Gene

Genomic localization for the hElk gene was determine by amplifying theStanford Genome Center's G3 Radiation Hybrid panel with the followinghElk specific oligonucleotides:

CACCTGGGATGGCTTCATCCTGCTC (SEQ ID NO:7) AAACACCACCTGGCCCGACTTGGAC (SEQID NO:8)

hElk was found to be linked to marker SHCG-33198 with an LOD score of6.075282, placing the hElk gene approximately 100 kbp from this marker(e.g., 4.71 millirads). This marker is on chromosome 12 and appears tobe in the 12pter-12qter band near 12q13. The Gene Location database mapsa nearby marker (SHGC-30065) at 52.32 centimorgans from the top of thechromosome 12 recombination group. The closest mapping disease in thisgroup is Allgrove syndrome, which is a rare adrenal insufficiencyassociated with a collection of poorly defined neuropathies. AlthoughAllgrove appears to fall between markers that may exclude hElk, it ispossible that hElk maps to Allgrove.

1. An antibody that selectively binds to a polypeptide consisting of theamino acid sequence of SEQ ID NO:1, wherein the selective binding is atleast twice background signal.
 2. The antibody of claim 1, which is apolyclonal antibody.
 3. The antibody of claim 1, which is a monoclonalantibody.